PROJECT SUMMARY-ABSTRACT Human cytomegalovirus (HCMV) infection causes serious morbidity/mortality in immune-compromised patients and leads to high rates of birth defects after congenital infection. HCMV vaccine development is thus a public health priority. However, HCMV is one of the most intricate human viruses and unique in its broad-range infectivity in a wide range of human cells. Identifying conserved molecular targets is of critically importance for blocking the versatile infectivity of HCMV. Recently, we undertook an investigation to explore whether viruses of distinct phylogenetic origins, such as HCMV, human immunodeficiency virus 1 (HIV-1), and severe acute respiratory syndrome coronavirus (SARS- CoV), express conserved glyco-determinants that are suitable for broad-spectrum virus neutralization. We assumed that viruses depend on host glycosylation machinery for glycan synthesis and thereby may express the conserved viral carbohydrates. This study has led to identification of a number of cryptic glycan markers overexpressed by these viruses. Importantly, these carbohydrates carry potent virus-neutralizing epitopes and are specifically targeted by a broad-spectrum virus-neutralizing agent, Galanthus nivalis lectin (GNA). These findings prompt us to further investigate whether these carbohydrates, as displayed by HCMV, are immunogenic in vivo and thereby serve as key targets for immunological intervention against HCMV infection. Specifically, we will perform a cross-species carbohydrate microarray study to examine whether host immune systems recognize and mount neutralizing antibody responses to these targets (Aim 1). Moreover, we will take advantage of a large panel of anti-glycan monoclonal antibodies and lectins of defined specificities to pinpoint the dominant antigenic determinants that are targeted by broad-spectrum virus-neutralizing antibodies (Aim 2). [In summary, we propose two complementary approaches to identify potential immunogenic carbohydrate moieties of HCMV, which is a critical step for developing novel glyco-conjugate vaccines against HCMV.] The strength of this proposal is substantially enhanced by key collaborators with unique expertise in HCMV vaccine development, chemo-enzymatic synthesis of carbohydrate antigens, and HCMV healthcare in hospitals. Since the targeted carbohydrates are also overexpressed and surface-exposed by other viral pathogens, such as HIV-1, SARS-CoV, and the Ebola virus, this study may provide key information to guide the production of a cocktail of anti-glycan antibodies that are readily applicable for passive immunization against a broad range of viral pathogens.